Millul V, Ardaillou N, Placier S, Baudouin B, Ronco P M
INSERM U64, Hôpital Tenon, Paris, France.
Kidney Int. 1997 Jan;51(1):281-7. doi: 10.1038/ki.1997.34.
The aim of the present study was to analyze the expression of natriuretic peptide receptors in human collecting duct, by using a newly established SV40 cell line (HCD). ANP and C-type natriuretic peptide (CNP) induced a concentration-dependent increase in cGMP suggesting the presence of type-A (NPR-A) and type-B (NPR-B) receptors, respectively. Threshold concentrations were 1 pM and 1 nM, respectively, and stimulated over basal cGMP ratios were 500 and 160 at 0.1 microM ANP and CNP. The urodilatin concentration-response curve was similar to that of ANP. [125I]-ANP bound specifically to HCD cells in a time-dependent fashion, reaching a plateau-phase between one and two hours at 4 degrees C. Equilibrium saturation binding curves suggested a single group of receptor sites (Kd = 421 +/- 55 pM, Bmax = 49.2 +/- 8.8 fmol/mg protein, Hill coefficient = 1.44 +/- 0.1, N = 6). Binding of [125I]-ANP was not displaced by CNP or by C-ANP (4-23), a specific ligand of clearance receptors (NPR-C), and thus occurred mainly via NPR-A. Neither Northern blot analysis nor RT-PCR could detect NPR-C mRNA, although the latter was clearly identified in control human glomerular visceral epithelial cells. In contrast, PCR products with the expected lengths were obtained for NPR-A and NPR-B. In conclusion, HCD cells express both NPR-A and NPR-B, as demonstrated by mRNA and cGMP production studies, but fail to produce NPR-C. This suggests that the human cortical collecting duct is a target for ANP, CNP and urodilatin.
本研究旨在通过使用新建立的SV40细胞系（HCD）来分析人集合管中利钠肽受体的表达。心房钠尿肽（ANP）和C型利钠肽（CNP）可诱导cGMP浓度依赖性增加，分别提示存在A型（NPR-A）和B型（NPR-B）受体。阈值浓度分别为1 pM和1 nM，在0.1 μM ANP和CNP作用下，相对于基础cGMP的刺激倍数分别为500和160。尿舒张素的浓度-反应曲线与ANP相似。[125I]-ANP以时间依赖性方式特异性结合HCD细胞，在4℃下1至2小时达到平台期。平衡饱和结合曲线提示存在单一组受体位点（Kd = 421±55 pM，Bmax = 49.2±8.8 fmol/mg蛋白，希尔系数 = 1.44±0.1，N = 6）。[125I]-ANP的结合不会被CNP或C-ANP（4-23）（清除受体（NPR-C）的特异性配体）取代，因此主要通过NPR-A发生。Northern印迹分析和逆转录-聚合酶链反应（RT-PCR）均未检测到NPR-C mRNA，尽管在对照人肾小球脏层上皮细胞中可明确鉴定出后者。相反，获得了预期长度的NPR-A和NPR-B的PCR产物。总之，如mRNA和cGMP生成研究所证明的，HCD细胞同时表达NPR-A和NPR-B，但不产生NPR-C。这表明人皮质集合管是ANP、CNP和尿舒张素的作用靶点。
